The avidin-biotin system has been known for many years as an exceptional tool for qualitative and quantitative studies in interactions between small molecules and biological receptors (Wilchek, M., et al., Immunol. Today, 1984, 6, 39).
Avidin is a glycoprotein of about 68 kDa present in the egg white and showing high affinity for the vitamin H biotin. Its dissociation constant (Kd˜10−15M) is the lowest known in nature (Green N. M., et al., Biochem. J., 1970, 118, 67; Green, N. M., Adv. Protein Chem., 1975, 29, 85). It is composed of four subunits of identical amino acid sequence, each of which can potentially bind one molecule of biotin. Glycosylation accounts for about 10% of its molecular weight with an average of four to five mannose and three N-acetylglucosamine residues per subunit (Bruch R. C., et al., Biochemistry, 1982, 21, 5334).
In 1988, a study regarding interaction between radiolabelled biotin derivatives and avidin was reported (Garlick R. K., et al., J. Biol. Chem., 1988, 263, 210).
In WO04093916 in the name of the Applicant, a two-step perioperative therapy of solid tumours was described as a novel form of brachytherapy. The first step involved the administration within the operated area of a biotinylated specific antibody followed by injection of native or PEG-ylated avidin in order to construct an “artificial receptor”. Then, within the second step, the proper anticancer agent coupled to biotin was administered systemically. The second step needed to be performed within 4 to 72 hours from the surgical removal of the tumour. However, no suggestions of direct avidination through covalent binding of avidin to the considered tissue were provided.
Clinical applications of this two-step brachytherapy using avidin in the first step and radiolabelled biotin-DOTA (ST2210) in the second step, proved to be effective in delivering partial irradiation to the surgically operated area in breast cancer patients (Paganelli G., et al., The Breast, 2007, 16, 17; Paganelli G., et al., Clin. Canc. Res., 2007, 13, 5646). The radiation dose released to the surgically operated index quadrant, in 11 patients, was an average of 20 Gy for an administered dose of 100 mCi. This boost represents about ⅓ of the expected 60 Gy delivered to this type of patients by the current standard External Beam Radiotherapy (EBR).
The uses of streptavidin antibody constructs with biotin-radionuclide conjugates in the treatment of patients with malignant gliomas, and of bispecific antibodies with hapten-radionuclides in the therapy of tumours expressing carcinoembryonic antigen were reported recently (Goldenberg, D. M., et al., J. Clin. Oncol., 2006, 24, 823). However, in quite a few cases renal toxicity appeared due to the too elevated dose that passed the kidney.
One of the main problems when treating with avidin, resides in its rapid clearance from the body. Lately, research efforts focussed at finding “modified avidin” with a longer half-life. One such approach consisting in linking the protein via free amino groups to monomethoxypolyethylene glycol, resulted in prolonged plasma half-life of the modified avidin with 8% of the i.v. injected dose still present in the tumour after 5 hours and 6% after 72 hours when avidin was coupled to PEG-20 kDa (Caliceti P., et al., J. Control. Release, 2002, 83, 97).
A pharmacokinetic study demonstrating the influence of the size of the PEG moiety highlighted the fact that the heavier the PEG unit the shorter the half-life, meanwhile biotin-avidin affinity degree was following an opposite trend (Salmaso S., et al., Biochim. Phys. Acta, 2005, 1726, 57).
Another pharmacokinetic and biotin-binding properties study on different PEG-ylated avidin showed that 7 PEG moieties per avidin protein was the best ratio, allowing increasing the plasma half-life and reducing the immunogenecity of avidin. However, no details were given in animal model with regard to biotinylated drug accumulation within tumours (Chinol M., et al., Br. J. Cancer, 1998, 78, 2, 189).
As an alternative to PEG-avidin, thermoresponsive polymers have been investigated. Poly(N-isopropylacrylamide-co-acrylamide)-avidin showed higher residence time in bloodstream compared to avidin and lower accumulation within the liver (Salmaso S., et al., Int. J. Pharm., 2007, 340, 20). However, also in this case no details in animal model with regard to biotinylated drug accumulation within tumours were reported.
Unfortunately, up to now, no efficient and selective method to specifically localize therapeutic agents is available.
Therefore, improvement of anticancer therapy is still a great need and a major area of efforts for pharmaceutical companies.
Avidin-biotin binding interaction is dependent on the protein portion. In fact, deglycosylated avidin preserves the biotin binding capacity (Hiller Y., et al, Biochem. J., 1987, 248, 167; Rosebrough S. F., et al., J. Nucl. Med., 1996, 37, 8, 1380).
Increased accumulation and permanence of a therapeutic agent within the area to be treated could be achieved through the avidination of the tissue with a modified avidin endowed with a higher tissue permanence compared to wild type avidin.
Such a strategy would avoid the need of systemic administration of avidin and consequently prevent any side effects related to such therapy. In addition, an increased avidination of the relevant tissue would result in a less toxic treatment due to lower distribution of the anticancer agent in non relevant organs and reduced dose of anticancer agent to obtain the same effect as with the wild type avidin.
It has now been found that by oxidizing the glycosylated part of avidin, stable avidination of surrounding tumour tissue can be obtained, allowing a biotinylated anticancer agent to better concentrate in such region.